1. Field of the Invention
The present invention relates to a hemopoietic growth-regulatory calmodulin-binding protein and methods of using the same in the management of both non-malignant and malignant hematological diseases.
2. Description of the Background
Proliferation of eukaryotic cells is controlled by a series of regulatory events, including expression and intracellular redistribution of enzymes and other proteins associated with DNA synthesis, occurring during G1 phase of the cell cycle. Although it is well known that growth factors or cytokines are required to stimulate the proliferation of hemopoietic cells, very little is known about the regulatory process(es) stemming from growth factor/receptor interactions on the membrane that culminates in the induction of nuclear DNA replication and cellular proliferation.
Calmodulin (CAM) and other members of the family of calcium-modulated proteins are major eukaryotic intracellular receptors for the element calcium, a substance of major importance in the regulation of many diverse cell processes such as motility, proliferation and nutrient utilization.
Calmodulin is ubiquitously distributed in eukaryotic cells and has been isolated from bovine brain, rat testis and marine invertebrates. The protein is a relatively small, acidic, stable protein having a molecular weight of 15,000 to 19,000 lacking cysteine, hydroxyproline and tryptophan. Moreover, it has a high content of acidic amino acids and low tyrosine content and almost all calmodulins isolated contain a single, fully trimethylated lysyl residue.
Further, calmodulins obtained from a wide variety of phylogenetically different sources are similar in amino acid sequence and in physico-chemical and biological properties. Hence, the protein lacks both species and tissue specificity and appears to be structurally and functionally conserved throughout evolution.
Progression of eukaryotic cells from G1 to S phase is also known to be highly dependent on events regulated by calcium, which is known to exert its effects either by controlling Ca.sup.2+ -sensitive, phospholipid-dependent protein kinase C, or activating the Ca.sup.2+ -binding protein, CaM. Both of these pathways are induced by the interaction of extracellular growth factors with their membrane receptors which stimulate a membrane-bound enzyme, phospholipase C. Phospholipase C catalyzes the conversion of phosphatidylinositol 4,5-bisphosphate to diacylglycerol or to inositol 1,4,5-triphosphate. Diacylglycerol activates phosphatidyl serine-dependent protein kinase C which phosphorylates serine and/or threonine residues of its target proteins. Inositol 1,4,5-triphosphate is believed to release Ca.sup.2+ from the endoplasmic reticulum stores, leading to the activation of CaM. Activated CaM regulates cellular cyclic nucleotides by activating phosphodiesterase, adenylate cyclase and guanylate cyclase, and protein phosphorylation through activation of CaM-dependent protein kinases and phosphatases. At present, the involvement of protein kinase C and cyclic nucleotides in cellular proliferation is unclear. Further, the role of CaM, activated by inositol 1,4,5-triphosphate-dependent release of Ca.sup.2+, also remains obscure
Inositol 1,4,5-triphosphate is known to stimulate initiation of DNA synthesis by releasing Ca.sup.2+ from intracellular stores and Ca.sup.2+ channels are known to be involved in signal transduction of hemopoietic growth factors. By lowering Ca.sup.2+ levels in cellular medium at the late-prereplicative period (G1/S boundary) of mammalian cells, it appears possible to prevent cells from initiating DNA synthesis by stopping the expression and/or activation of ribonucleotide reductase, and the synthesis of four deoxynucleotides. Further, Ca.sup.2+ -deprived cells appear to undergo dismantling of prereplicative structure and enter a state of quiescence.
Since most of the effects of Ca.sup.2+ are known to be mediated by the activation of CaM, changes in intracellular CaM levels can also govern Ca.sup.2+ -mediated events. In fact, the integrated role of Ca.sup.2+ and cellular proliferation is strongly implicated from the following observations: a) CaM levels are elevated 2 to 3 fold at the G1/S boundary; b) there is a complete coincidence between temporal increase in CaM levels and the progression of cells into S phase, regardless of the length of G1; c) there is a direct correlation between intracellular CaM levels and the ability of cells to replicate DNA; d) CaM can stimulate DNA replication in isolated liver cells; e) nuclear CaM undergoes rearrangement during proliferative activation of liver cells; f) progression of cells from G1 to S phase is sensitive to CaM antagonists; and g) the stimulatory effect of Ca.sup.2+ on DNA synthesis in rat hepatocytes is mimicked by the addition of CaM and is blocked by CaM antagonists and anti-CaM antibody. Thus, CaM, in serving as a Ca.sup.2+ receptor, appears to play a critical role in the control of cell proliferation.
However, while the interaction between hemopoietic growth factors and their cognate receptors is known to be the primary event in hemopoiesis, the molecular events in proliferative signal transduction following ligand-receptor interaction are not clear. Moreover, at present, there is no known means by which CaM activity can be controlled in order to control the onset of DNA synthesis. It would be extremely desirable, however, if such a means could be found for controlling the progression of cells from G1 into S phase of the cell cycle. Such a means would make it possible to manage both non-malignant and malignant hematological diseases.